Malate thiokinase catalyzes an ATP dependent thiol ester formation between Coenzyme A and a number of dicarboxylic acids. The reaction is analogous to the well known succinate thiokinase reaction. Upon purification of malate thiokinase using 3'-5' AMP coupled to Sepharose 4B, two active forms of the enzyme have been observed as judged by electrophoresis on polyacrylamide gels. One form has been identified as the native form of the enzyme, mol wt approximatily 180,000 and composed of 4 alpha and 4 beta subunits. The other active form of the enzyme is produced by an interaction of succinyl CoA with the enzyme, has a molecular weight of approximately 100,000 and apperas to be composed of 2 alpha and 2 beta subunits. The S-form of the enzyme can be converted back to the N-form by reaction with ATP or which ADP plus Pi. The S-form of the enzyme appears to be the catalytically active form. Displacement of bound succinyl CoA results in the concomittant formation of phospho-enzyme. The data suggests that malate thiokinase can be classified as an enzyme exhibiting half-of-the-sites reactivity. An obligatory alternating site mechanism does not appear to be applicable to this enzyme.